Recent efforts have been given to measurement techniques for determining the presence, and possibly the level of concentration, of targets in a larger mixture or solution in which the particles reside. It is often desirable to measure relatively low concentrations of certain organic compounds. In medicine, for example, it is very useful to determine the concentration of a given kind of molecule, usually in solution, which either exists naturally in physiological fluids e.g. blood or urine or which is introduced into the living system e.g. drugs.
One broad approach used to detect the presence of a particular compound of interest is the immunoassay technique, in which detection of a given molecular species, referred to generally as the ligand, is accomplished through the use of a second molecular species, often called the antiligand or the receptor, which specifically binds to the ligand of interest. The presence of the ligand of interest is detected by measuring, or inferring, either directly or indirectly, the extent of binding of ligand to antiligand e.g. by optical methods. A ligand can be considered a target or an analyte.
There are a number of immunoassay formats. All involve bindings but not all involve agglutination. In the non-agglutinating case, one label is generally attached to one target. Agglutination assays are rapid and easy to detect, and they are used when easy detection and immediate results are required e.g. in the field.
Quantitative tests based on optical detection of large particles are only moderately sensitive because they rely on measurements of turbidity (transmitted light through sample) or nephelometry (scattered light through sample), both of which are influenced by background interference from particulate matter. Consequently, optical methods are not suitable for use with raw samples such as whole blood, which contains cells, and saliva, which may have food particles.
Instead, magnetic particles made from magnetite and inert matrix material has long been used in the field of biochemistry. They range in size from a few nanometers up to a few microns in diameter and may contain from 15% to 100% magnetite. They are often described as superparamagnetic particles or, in the larger size range, as magnetic beads. The usual methodology is to coat the surface of the particles with some biologically active material which will cause them to bind strongly with specific microscopic objects or particles of interest e.g. proteins, viruses, cells, or DNA fragments. The magnetic particles may be considered as “handles” by which the objects can be moved or immobilized using a magnetic gradient, usually provided by a strong permanent magnet.
Previously, such magnetic particles have been used primarily for immobilizing the bound objects, but recent work has been done on using the particles as tags for detecting the presence of the bound complexes. Historically the detection and quantification of the bound complexes have been accomplished by means of radioactive, fluorescent, or phosphorescent molecules which are bound to the complexes of interest. However, these prior tagging techniques have various well-known disadvantages.
On the other hand, since the signal from a tiny volume of magnetic particles is exceedingly small, it has been natural that researchers have tried building detectors based on Superconducting Quantum Interference Devices (SQUIDs), which are well known to be the most sensitive detectors of magnetic fields for many applications. However, SQUIDs are quite sensitive measurement devices, but suffer inter alia from the disadvantage that the devices have to be cooled around cryogenic temperatures.
Recently, improved magnetic particle sensor devices have been disclosed by the present applicant, in particular in international patent applications WO 2005/010542 and WO 2005/010543, which are both hereby incorporated by reference in their entirety. These magnetic particle sensor devices have the advantages that measurement can be performed at around room temperature while at the same time having a sufficiently high signal-to-noise ratio (SNR).
U.S. Pat. No. 6,437,563 (to Simmonds et al. and Quantum Design, Inc.) recently disclosed an apparatus, which is provided for quantitatively measuring combinations of magnetic particles combined with analytes whose amount or other characteristic quality is to be determined. The magnetic particles are complexed with the analytes to be determined and are excited in a magnetic field of several hundred kHz. The magnetizations of the magnetic particles are thereby caused to oscillate at the excitation frequency in the manner of a dipole to create their own fields. These fields are inductively coupled to at least one sensor such as sensing coils fabricated in a gradiometer configuration. The output signals from the sensing coils are appropriately amplified and processed to provide useful output indications for combinations or agglutination of magnetic particles. However, working in the kilo-Hertz regime of oscillation may introduce unnecessary noise contributions, and additionally the application of a moving, in particular rotating, sample holder complicates the design as the rotating sample holder has to be relatively precise when the rotation is applied for decoupling of measurement and excitation. Additionally, the rotating sample holder does not easily facilitate sample manipulation immediately prior to, during, or immediately after magnetic measurement. Moreover, the coil technology applied by Simmonds et al. is not very sensitive, and thus relatively large amount of magnetic material is required for detection, which therefore increases the need for higher sample volumes. Finally, Simmonds et al. only measures the total amount of magnetic material both in the form of clusters and single particles, and effectively they do not distinguish between the two forms making it impossible to measure agglutination parameters.
It is also known that magnetic particles can rotate in a rotating magnetic field up to a certain frequency, the so-called critical slipping frequency. Above the critical slipping frequency the physical rotation of the magnetic particle cannot follow the rotations of the applied magnetic field.
Although a number of techniques for measuring of agglutination parameters exist, there is need for a more efficient and/or more reliable and/or more sensitive method. For example, it is a challenge in agglutination assays to detect a low number of clustered species in a background of many un-clustered species, and/or to detect a low number of un-clustered species in a background of many clustered species.